Mutations In Mitochondrial tRNA Genes Research Articles

BCL-2 Improves Oxidative Phosphorylation and Modulates Adenine Nucleotide Translocation in Mitochondria of Cells Harboring Mutant mtDNA

Members of the BCL-2-related antiapoptotic family of proteins have been shown previously to regulate ATP/ADP exchange across the mitochondrial membranes and to prevent the loss of coupled mitochondrial respiration during apoptosis. We have found that BCL-2/BCL-x(L) can also improve mitochondrial oxidative phosphorylation in cells harboring pathogenic mutations in mitochondrial tRNA genes. The effect of BCL-2 overexpression in mutated cells was independent from apoptosis and was presumably associated with a modulation of adenine nucleotide exchange between mitochondria and cytosol. These results suggest that BCL-2 can regulate respiratory functions in response to mitochondrial distress by regulating the levels of adenine nucleotides.

Molecular investigations on tRNAs involved in human mitochondrial disorders.

Over the last decade, human neurodegenerative disorders which correlate with point mutations in mitochondrial tRNA genes became more and more numerous. Both the number of mutations (more than 70) and the variety of phenotypes (cardiopathies, myopathies, encephalopathies as well as diabetes, deafness or others) render the understanding of the genotype/phenotype relationships very complex. Here we first summarize the efforts undertaken to decipher the initial impact of various mutations on the structure/function relationships of tRNAs. This includes several lines of research, namely (i) investigation of human mitochrondrial tRNA structures, (ii) comparison of disease-related and polymorphic mutations at a theoretical level, and (iii) experimental investigations of affected tRNAs in the frame of mitochondrial protein synthesis. A new approach aimed at searching for long-range effects of mitochondrial tRNA mutations on a broader global mitochondrial level will also be presented. Initial results obtained by comparative mitochondrial proteomics turn out to be very promising for deciphering unexpected molecular partners involved in the pathological status of the mitochondria.

A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(Ile).

Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies. We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon. As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(Ile) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides. We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(Ile) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(Ile) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(Ile) itself is intrinsically unstable.

Suppression of a mitochondrial tRNA gene mutation phenotype associated with changes in the nuclear background

We previously have characterized a pathogenic mtDNA mutation in the tRNAAsn gene. This mutation (G5703A) was associated with a severe mitochondrial protein synthesis defect and a reduction in steady-state levels of tRNAAsn. We now show that, although transmitochondrial cybrids harboring homoplasmic levels of the mutation do not survive in galactose medium, several galactose-resistant clones could be obtained. These cell lines had restored oxidative phosphorylation function and 2-fold higher steady-state levels of tRNAAsn when compared with the parental mutant cell line. The revertant lines contained apparently homoplasmic levels of the mutation and no other detectable alteration in the tRNAAsn gene. To investigate the origin of the suppression, we transferred mtDNA from the revertants (143B/206 TK-) to a different nuclear background (143B/207 TK-, 8AGr). These new transmitochondrial cybrids became defective once again in oxidative phosphorylation and regained galactose sensitivity. However, galactose-resistant clones could also be obtained by growing the 8AGr transmitochondrial cybrids under selection. Because the original rate of reversion was higher than that expected by a classic second site nuclear mutation, and because of the aneuploid features of these cell lines, we searched for the presence of chromosomal alterations that could be associated with the revertant phenotype. These studies, however, did not reveal any gross changes. Our results suggest that modulation of the dosage or expression of unknown nuclear-coded factor(s) can compensate for a pathogenic mitochondrial tRNA gene mutation, suggesting new strategies for therapeutic intervention.

Impairment of tRNA processing by point mutations in mitochondrial tRNA Leu(UUR) associated with mitochondrial diseases

Several point mutations in mitochondrial tRNA genes have been linked to distinct clinical subgroups of mitochondrial diseases. A particularly large number of different mutations is found in the tRNA Leu(UUR) gene. We show that base substitutions at nucleotide position 3256, 3260, and 3271 of the mitochondrial genome, located in the D and anticodon stem of this tRNA, and mutation 3243 changing a base involved in a tertiary interaction, significantly impair the processing of the tRNA precursor in vitro. In correlation with other studies, our results suggest that inefficient processing of certain mutant variants of mitochondrial tRNA Leu(UUR) is a primary molecular impairment leading to mitochondrial dysfunction and consequently to disease.

Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae.

Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated. Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345). The latter mutation clearly affects the 3' end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation. The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA. These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation.

Point mutations in mitochondrial tRNA genes: sequence analysis of chronic progressive external ophthalmoplegia (CPEO)

We have sequenced all mitochondrial tRNA genes from 9 Japanese patients with chronic progressive external ophthalmoplegia (CPEO) who had no detectable large mtDNA deletions nor mutations previously reported, and identified 6 different base substitutions in 6 patients. Since 5 of the 6 substitutions were homoplasmic in distribution and recognizable in some normal controls, they were thought to be polymorphisms in normal individuals. One mutation at nucleotide (nt) 12311 in the tRNA Leu( cun) gene was not present in 90 normal controls nor in 103 patients with other mitochondrial myopathies. This mutation was in a heteroplasmic state, and the mutated site was conserved among other species during evolution, suggesting a disease-related mutation. However, the significance of this mutation has to be studied further. In Japanese CPEO patients without large deletions, a point mutation in the mitochondrial tRNA gene is not likely to be a frequent cause.

Mutations in mitochondrial tRNA genes: non-linkage with syndromes of Wolfram and chronic progressive external ophthalmoplegia.

We have recently identified a point mutation in the mitochondrially encoded tRNA(Leu(UUR)) gene which associates with a combination of type II diabetes mellitus and sensorineural hearing loss in a large pedigree. To extend this finding to other syndromes which exhibit a combination of diabetes mellitus and hearing loss we have sequenced all mitochondrial tRNA genes from two patients with the Wolfram syndrome, a rare congenital disease characterized by diabetes mellitus, deafness, diabetes insipidus and optic atrophy. In each patient, a single different mutation was identified. One is an A to G transition mutation at np 12,308 in tRNA(Leu(CUN)) gene in a region which is highly conserved between species during evolution. This mutation has been described by Lauber et al. (1) as associating with chronic progressive external ophthalmoplegia (CPEO). The other is a C to T transition mutation at np 15,904 in tRNA(Thr) gene. Both mutations are also present in the general population (frequency tRNA(Leu(CUN)) mutation 0.16, tRNA(Thr) mutation 0.015). These findings suggest that evolutionarily conserved regions in mitochondrial tRNA genes can exhibit a significant polymorphism in humans, and that the mutation at np 12,308 in the tRNA(Leu(CUN)) gene is unlikely to be associated with CPEO and Wolfram syndrome.

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.

The mapping of mutations in tRNA and cytochrome oxidase genes located in the cap-par segment of the mitochondrial genome of S. cerevisiae

1. Two mutants have been isolated which carry mutations in mitochondrial tRNA genes; one in the aspartyl-tRNA gene and the other in one of the threonyl-tRNA genes. The mutant tRNAs are unable to be charged with their respective amino acids. 2. These two mutations are located on the mitochondrial genome in the cap-par segment. Analyses of genetic recombination frequencies and co-retention and co-deletion frequencies of markers in petite strains yield an unambiguous gene order cap-asp-oxi 1-thr 1-oxi 2-par. 3. Two loci involved in the specification of cytochrome oxidase (oxi1 and oxi2) show an average recombination frequency of 14% in pairwise crosses involving mutations of both loci. Although the two loci have a related function and are genetically linked they are shown to be separated by at least one tRNA gene. 4. Pairwise intralocus crosses involving mit- mutations within either the oxi1 or oxi2 locus yield recombination frequencies <0.02–3.2%. However, no unique order could be derived for 19 oxi 1 and 6 oxi 2 mutations based on these data. In addition, the separation of mutant alleles within a single locus as the result of petite mutation was very rare. Consequently, attempts to order mutations within the locus from an analysis of the flanking markers in petites where separation occurred, provided only limited resolution. 5. A discussion is presented of the limitations of the current mitochondrial genetic mapping techniques when applied to the fine resolution of glycerol negative mit- and syn- mutations.